Chronic undernutrition resulting in significant weight retardation and linear growth failure has long been recognized as a general problem among cystic fibrosis patient populations, as well as those resulting from other diseases or syndromes of malabsorption of fat from the small intestine. It is well accepted in medical science that there is a significant correlation between the degree of malnutrition and the severity of such diseases. A variety of complex factors, both related and unrelated, can give rise to the energy imbalance underlying undernutrition or malnutrition that manifests in malabsorptive conditions. Nonetheless, fecal nutrient losses, including those of fat, from maldigestion or malabsorptive diseases are known to contribute to energy imbalance, and therefore point to the need for a convenient and accurate measure of fat absorption to improve diagnosis, management, and treatment of these conditions. While the normal value for total fat absorption in humans is >90% absorbed (<10% remaining in feces), it may be as low as 60% (40% remaining in feces) in association with conditions of atrophy of intestinal mucosa such as celiac disease, idiopathic steatorrhea, obstructive jaundice, chronic pancreatitis, cystic fibrosis, gastrectomy; intestinal resection or anomalies, blockage of intestinal lymphatics, iatrogenic steatorrhea caused by irradiation or antibiotics, pneumatosis intestinalis, or failure of blood supply such as mesenteric endarteritis.
Fat-balance methodology, which is based on the measurement of consumed and excreted fat, is a standard means for the assessment of the absorption of dietary fat. Fat-balance methodology can be used for indirect measurement of fat absorption because fat that is not absorbed in the small intestine is minimally altered during transit through the large intestine. Although hydrolysis of dietary triacylglycerol fats can be catalyzed by anaerobic bacterial lipases in the large intestine, long-chain fatty acids released from triacylglycerol fats are not utilized for energy in the colon and are excreted intact or with structural alterations limited to partial hydrogenation or migration of double bonds (Howard, F A et al. (1999). Lett Appl Microbiol 29:193-196). These alterations do not measurably affect the mass of unabsorbed fat that appears in feces. Therefore fecal fat reflects the type and amount of fat that exits the small intestine.
Although the basic concept of “fat intake minus fat output” is easily understood, the execution of the fat-balance method is difficult in practice. First of all, an accurate and complete measurement of diet composition and consumption is necessary for the calculation of fat absorption. Studies with rodents can be problematic if diet is spilled or scattered, thus making it difficult to determine the amount of diet that was consumed. Equally important is the complete collection of fecal matter at a time and of a duration corresponding to the test meals. This collection can require special metabolic cages for animal studies. Methods to help match fecal matter with corresponding test meals have included radio-opaque pellets in the diet with subsequent counting of the pellets in rodent feces.
Accurate and complete collection of human fecal matter is more problematic, and can involve a stay of 4-7 days in a metabolic ward. Current methodology is based on the assumption that all unabsorbed fat ingested from a test meal has been recovered in the collection of fecal material. Typically, all feces excreted during a 72-hour period following ingestion of a test meal are collected. Such a collection can be difficult to execute accurately, and can be an onerous task, as well as difficult to verify as complete. The difficult nature of the complete collection of fecal matter for clinical analysis can be more fully understood by the following description of the methodology taken from Bray's Clinical Laboratory Methods (CV Mosby Co., Library of Congress Catalog Card Number 68-55316, pg. 455), which represents the state of the art in collection of samples for fecal fat analysis: “Collection of the specimen presents some problems. The determination of fat on a random specimen is of little value. It is generally agreed that, if possible, all the stool excreted over a 3-5 day period should be collected for analysis. Also it is preferable that the patient be on a fairly constant diet, one in which the fat content is at least approximately known, for 2 or 3 days prior to and throughout the collection period. The samples should be preserved in the refrigerator until analyzed. If more than an occasional determination is made (i.e., multiple subjects are to be tested), it is helpful to collect the specimens in new pre-weighed 1-gallon metal paint cans. These have tight-fitting covers, and after the entire specimen has been collected it can be well mixed in the original can by adding some water if necessary and shaking on a paint-shaking machine. This usually gives a homogeneous sample. Subtract the weight of the can from the weight of the can plus contents to obtain the weight of the specimen. The addition of water makes no difference since one is determining the fat in an aliquot from an entire 3-day specimen. If the feces are collected in other containers, the entire specimen must be well mixed. This is best accomplished with a Waring blender or similar machine and with the addition of a small amount of water. The weight of the total homogenized specimen must then be obtained.”
Because of the problems associated with complete collection of fecal specimens, other techniques have been used to estimate fat absorption (Hill, P G, (2001). Ann. Clin. Biochem. 38:164-167). For example, the measurement of the appearance of 14CO2 in breath after consumption of 14C-triolein has been carried out. This approach gives a relative measure of absorption based on comparison with 14CO2 in the breath of subjects for which fractional fat absorption is known. In a modification of this method to avoid exposure to radioactivity, dietary triacylglycerols containing 13C have also been fed followed by subsequent measurement of expired 13CO2 by mass spectroscopic analysis. Non-absorbable flow markers have also been used in the measurement of dietary lipid absorption. Cholesterol absorption has been measured by radioisotope techniques using dietary plant sterols as non-absorbable fat markers (see e.g., Jandacek, R J et al. (1990). Metabolism, 39:848-852). Dietary triacylglycerol absorption has been measured in rats by simultaneous feeding of 133I-triolein and the non-absorbable fat marker, 75Se-glyceryl triether (Hoving, J. et al. (1977). Gastroenterology 72:406-412). However, these markers obviously require ingestion of radioactive material, a procedure that has inherent limitations, and can have serious drawbacks for human subjects.
Non-radioactive safe markers that are measured by standard techniques have not been validated for use in measuring fat absorption. Currently, there are neither any known markers that are both safe and readily available for use in humans, nor methods for the use of use such a marker. Therefore, a need exists for an appropriate marker for dietary fat that: (1) is not absorbed; (2) has the physical properties of dietary triacylglycerol fats; (3) can be measured by standard gas chromatographic techniques; (4) is approved for use in humans, and (5) does not alter dietary fat absorption. Such a marker would also need to be readily available in sufficient, cost-effective quantities. Additionally, a method for measurement that did not require extensive collection and homogenization of fecal material would provide a significant advantage to both the subject and the administrator of the method. Therefore, a method that would allow measurements to be taken from relatively small samples taken at appropriate times would relieve all parties involved of the burden of extensive collection and storage of fecal materials.